1. Field of the Invention
This invention relates to a novel promoter for tissue specific gene expression and uses thereof. More particularly, this invention relates to nucleic acid sequences containing a functional promoter for the VEGF receptor, Flt-1 (fms-like receptor tyrosine kinase) that correspond in structure and/or properties to the native genomic form of this promoter, the use of such nucleic acid sequences in screening for drugs that affect the VEGF/Flt-1 regulatory pathway, the use of such nucleic acid sequences in endothelial specific gene expression and other therapeutic and diagnostic applications.
2. Background
The establishment of a vascular supply is a critical requirement for the cellular inflow of nutrients, outflow of waste products and gas exchange in most tissues and organs. Two separate processes for such blood vessel development and differentiation have been identified. One process, termed "vasculogenesis" takes place in the embryo and consists of the in situ differentiation of mesenchymal cells into hemoangioblasts which are the precursors of both endothelial cells and blood cells. The other process, termed "angiogenesis" is the formation of new blood vessels by sprouting from a preexisting endothelium. This process is required not only for the further development of the embryonic vasculature, but also for a wide variety of post natal processes such as wound healing and tissue and organ regeneration. In addition, angiogenesis has been identified as a critical requirement for solid tumor growth and uncontrolled blood cell proliferation is an important pathogenic component in a variety of other disorders such as rheumatoid arthritis, atherosclerosis, diabetes mellitus, retinopathies, psoriasis and retrolental fibroplasia (all of which are characterized by excessive angiogenesis). Therefore, there is much interest in identifying angiogenesis factors and their receptors, identifying their mechanisms of action with the goals of agonizing or antagonizing their activity and using their activity as a prognostic predictor of disease state.
Recently, vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) has been identified as a prime regulator of normal and pathological angiogenesis. VEGF is a secreted growth factor which has the following properties: (1) it is a remarkably specific mitogen for endothelial cells; (2) it is angiogenic in vivo and induces vascular permeability; (3) expression of VEGF and its receptors correlates with vasculogenesis and angiogenesis during embryonic development; and (4) VEGF is expressed in tumor cells, whereas the VEGF receptor is expressed exclusively in adjacent small blood vessels. VEGF plays a crucial role for the vascularization of a wide range of tumors including breast cancers, ovarian tumors, brain tumors, kidney and bladder carcinomas, adenocarcinomas and malignant gliomas. Tumors produce ample amounts of VEGF, which stimulates the proliferation and migration of endothelial cells (ECs), thereby inducing tumor vascularization by a paracrine mechanism.
The angiogenic effect of VEGF is mediated by its binding to high affinity cell surface VEGF receptors. Recently, three such high affinity receptors, Flt-1 (fms-like tyrosine kinase), Flk-1(fetal liver kinase-1; mouse homologue of kinase insert domain-containing receptor (KDR)) and Flt-4, have been cloned and identified. These receptors are members of the type III subclass of the family of tyrosine kinases. The type III subclass is characterized by proteins containing seven immunoglobulin-like domains, a single transmembrane region, and a kinase insert sequence. Of these, Flt-1 and Flk-1 are highly expressed by the endothelial cells in tumor blood vessels. In particular, high levels of Flt-1 expression are detected during periods of endothelial cell differentiation and neovascularization during wound healing and embryonic vascular development. Since endothelial cell differentiation drives tumor angiogenesis by promoting vascular permeability of the developing tumor blood vessels, inhibition of the production and activity of the VEGF/Flt-1 ligand/receptor system is a significant target for antiangiogenic anticancer strategies using anti-sense techniques, specific antibodies and specific inhibitors of VEGF/Flt-1 interactions.
Similarly, Flt-1 has been shown to be essential for the organization of the embryonic vasculature. Furthermore, Flt-1 is significantly up-regulated in a variety of disease states such as papillary dermal edemas, hemangioblastomas, cytokine-induced cell proliferation, CNS tumors and malignant gliomas. In comparison, there was little or no receptor expression in normal brain vasculature. Northern blot and in situ hybridization analysis showed significant Flt-1 mRNA transcription in capillary hemangioblastoma cells compared to normal brain cells. Therefore, it is believed that Flt-1 is induced during tumor progression and that the VEGF/Flt-1 signalling pathway plays a significant role in stimulating tumor angiogenesis, a requirement for solid tumor growth.
Though Flt-1 expression is greatly enhanced in diseased tissue such as solid tumors, little is known about the regulation of its expression. In addition, although as described earlier, Flt-1 expression is localized in endothelial cells, particularly in the vascular endothelium, little is known about its molecular regulation in the endothelium. Whereas the genomic DNA sequence of a related receptor tyrosine kinase containing two immunoglobulin-like domains, Tie, and its promoter region has been reported, the sequence of the promoter region of Flt-1 and other receptor tyrosine kinases of the type III subclass are not known. Knowing the promoter region of Flt-1 and other promoters of the type III subclass would enable the regulation of Flt-1 and the other type III subclass receptor tyrosine kinases. Regulation of Flt-1 expression would provide methods of inhibiting diseases associated with excessive angiogenesis of the vascular endothelium and promoting the processes of organ regeneration such as wound healing and the like. More generally, endothelial cells and the promoter elements controlling endothelial-specific gene expression are useful in the study of and therapy for diseases involving the vascular system, e.g., hemostasis, wound healing, atherosclerosis, hypertension, diabetic retinopathy, rheumatoid arthritis, blood cell trafficking, inflammatory conditions and tumor angiogenesis. Endothelial cells are in direct contact with blood and are therefore optimally situated for production of and secretion of desired proteins into the bloodstream or to neighboring cells. Expression systems where a gene is attached to an appropriate regulatory element targeted specifically to endothelial cells would allow for specific delivery of therapeutic agents to the endothelium. The novel promoters, nucleic acid sequences and screening assays provided by this invention fulfill these and other needs.
3. Summary of Related Art
Identification of the KDR Tyrosine Kinase as a Receptor for Vascular Endothelial Cell Growth Factor; B. Terman, M Dougher-Vermazen, M. Carrion, D. Dimitrov, D. Armellino, D Gospodarowicz, and P. Bohlen; Biochemical and Biophysical research Communications, V.187, No.3, 1579-1586, (1992); discloses cDNA and predicted amino acid sequence for KDR.
Nucleotide sequence and expression of a Novel Human Receptor-type Tyrosine kinase gene (flt) closely related to the fms family; M. Shibuya et al., Oncogene, 5, 519-524 (1990); discloses cDNA sequence of human Flt-1 gene.
The fms-Like Tyrosine Kinase, a Receptor for Vascular Endothelial Growth Factor; C. de Vries, J. Escobedo, H. Ueno, K. Houck, N. Ferrara, L. Williams; Science, 255, 989-991, (1992); describes cloning of human Flt-1.
Chromosomal Localization of FLT4, a Novel Receptor-Type Tyrosine Kinase Gene; F. Galland et al., Genomics, 13, 475-478, (1992); discloses the isolation, partial CDNA sequence and chromosomal localization of human Flt-4 and its deduced amino acid sequence.
Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth; K. Peters, C. De Vries, L. Williams; Proc. Natl. Acad. Sci. USA, 90, 8915-8919, (1993); discloses that Flt-1 expression was localized in the endothelium.
FLT4 Receptor Tyrosine Kinase Contains seven Immunoglobulin-like Loops and Is Expressed in Multiple Human Tissues and Cell Lines; K. Pajusola, O. Aprelikova, J. Korhonen, A. Kaipainen, L. Pertovaara, R. Alitalo, K. Alitalo; Cancer Research, 52, 5738-5743, (1992); discloses cDNA sequence for Flt-4.
Endothelial-Specific Gene Expression Directed by the tie Gene Promoter In Vivo; J. Korhonen et al., Blood, V.86, No.5, 1828-1835, (1995); discloses the human and mouse Tie gene promoter sequences.
Vascular Endothelial Cell lineage-specific Promoter in Transgenic Mice; Thorsten M. Schlaeger et al., Development, 121, 1089-1098 (1995); studied function of Tie-2 gene promoter in transgenic mice and embryonic stem cells.
Inhibitor of Vascular Endothelial Cell Growth Factor; R. Kendall, K. Thomas; PCT WO 94/21679, published Sep. 29, 1994; discloses soluble mutants of Flt-1 which bind VEGF with high affinity but do not result in signal transduction.
FLK-1 is a Receptor for Vascular Endothelial Growth Factor; PCT WO 94/11499, published May 26, 1994; discloses cloning of mouse Flk-1, uses of expressed Flk-1 to screen for drugs and inhibition of tumor growth using mouse cells encoding a transdominant-negative truncated Flk-1 mutant receptor.
Identification of a Novel Human Receptor Tyrosine Kinase Gene; PCT WO 92/14748, published Sep. 3, 1992; discloses the CDNA sequence of human KDR.